Sperm-egg fusion occurs at a critical moment in fertilization at which the zygote is formed. In recent progress key proteins on the gamete surfaces have been implicated in membrane fusion. Current evidence indicates: (1) sperm ADAMs may have a role in fusion and (2) the egg tetraspanin CD9 and egg GPI-anchored proteins are required for fusion. ADAM proteins potentially have either cell-cell adhesion activity or protease activity and we propose that each of these is required for normal fertilization. Although gene knockout studies have shown that ADAMs 1, 2 and 3 are not required for fusion, it is possible that other ADAM family members have a role. We will test if a different ADAM acts in fusion of the sperm and egg plasma membranes. We will also test if a known protease, ADAM 24, acts to create the membrane block to polyspermy. Since gene deletion studies have shown that egg CD9 is required for fusion, we focus on CD9's mechanism of action and its localization to lipid microdomains. CD9 is known to have a soluble ligand and we will test if sperm have a related transmembrane ligand to which CD9 binds. In tissue culture lines, CD9 associates with many other proteins on the surface of a CD9-expressing cell. We will test if an egg CD9-associated protein binds to the sperm surface. Gene knockout studies have also shown that egg GPI-anchored proteins are required for fusion. We propose to identify the critical GPl-anchored protein(s) and analyze its mode of action. Since multiple proteins appear to play a role in fusion, we ask how their activities are coordinated. We propose that to function in fusion the active egg molecules must be organized into lipid microdomains. Thus, these studies are designed to combine molecular and cellular levels to bring new insight into the process of gamete membrane fusion.